The objective of this proposal is to examine the relation between chromatin structure and transcriptional activity. In the past, studies on the biochemistry of RNA polymerase II transcription have generally employed naked DNA templates. While the use of naked DNA templates was expedient in terms of preliminary characterization of the factors, it is now apparent that many important aspects of transcription regulation must be analyzed in the context of chromatin rather than naked DNA templates. Current data suggest that the critical, early steps in gene activation involve alterations in chromatin structure that are mediated, at least in part, by transcription factors. In the proposed work, the transcriptional properties of reconstituted chromatin will be analyzed systematically. In addition, a combined in vivo and in vitro analysis of the chromatin will be analyzed systematically. In addition, a combined in vivo and in vitro analysis of the chromatin structure of a regulated gene will be performed. Specifically, the roles of nucleosomal cores, histone H1, HMG proteins, nucleosome positioning, and nucleosomal spacing upon regulation of RNA polymerase II transcription will be examined. These studies should lead to a better understanding of the process of gene activation and should eventually be applied to the molecular analysis of diseases involving improper expression of genes, such as some forms of cancer. In vitro transcription analysis with chromatin templates reconstituted from purified components. The aim of these studies is to perform a series of experiments with well-defined chromatin templates to examine the effects of specific components of chromatin (such as nucleosomal cores, histone H1, and HMG proteins) upon transcription by RNA polymerase II. Long range (greater than 1 kb) activation of transcription, such as the action of enhancers, will also be analyzed with the chromatin templates. Reconstitution and analysis of chromatin possessing physiological nucleosome spacing. The objective of these studies is to develop and to use a new chromatin reconstitution system that yields chromatin with physiological spacing of the nucleosomes. With the resulting chromatin, the effects of nucleosome spacing and nucleosome positioning upon transcription regulation will be examined. In addition, the determinants of nucleosome spacing during chromatin assembly will be investigated.